Abstract Prostate cancer (PCa) is the second most common cause of cancer-related death in men in the US, primarily due to the emergence of castration resistant prostate cancer (CRPC). Our long-term goal is to identify effective drug targets and develop small molecule inhibitors for CRPC. Our objective here is to determine the potential of LIMK2 kinase as a clinical target, and LI-11 (a highly potent and specific LIMK2 inhibitor) as a drug candidate for enzalutamide-resistant CRPC. The central hypothesis is that LIMK2 is a disease-specific target that is upregulated upon castration and promotes CRPC emergence, progression and enzalutamide resistance via upregulation of AR, ARV7 and PI3K pathways. We show that LIMK2 is highly expressed in CRPC tissues, but minimally expressed in normal prostates. Inducible knockdown or specific inhibition of LIMK2 fully reverses CRPC tumorigenesis in vivo, strongly supporting LIMK2 as a potential clinical target. Equally importantly, LI-11 synergistically enhances the efficacy of enzalutamide in CRPC cells. At a molecular level, LI-11 simultaneously depletes AR and ARV7, and inhibits PI3K signaling. Thus, LIMK2 targets three critical nodes driving CRPC progression including enzalutamide-resistance in tandem. We propose the following three specific aims: (1) Dissect the molecular mechanisms by which LIMK2 activates AR signaling in CRPC. (2) Investigate the molecular mechanism of LIMK2-mediated activation of PI3K pathway in CRPC. (3) Determine the potential of LIMK2 as a clinical target in enzalutamide-resistant CRPC and PDX models using LI-11 in vivo. Innovation: LIMK2 is a potential clinical target for PCa. The hypothesis was formulated based on an innovative chemical screen, which led to the discovery of several direct substrates of LIMK2. These LIMK2 substrates unlocked a powerful mechanism for therapeutic targeting of AR, ARV7 and PI3K pathways in tandem in CRPC. We have developed a highly potent and specific LIMK2 inhibitor (LI-11) that fully reverses tumorigenesis in vivo with no detectable toxicity. LI-11 will be used for preclinical validation of LIMK2 as a therapeutic target in cells and in vivo. LuCaP PDX models with variable LIMK2 levels will be used to determine whether LIMK2 levels correlate with LI-11 and enzalutamide sensitivity in vivo. Several CRISPR engineered cell lines will be utilized for genetic interrogation of LIMK2?s role in CRPC pathogenesis. Significance: Successful completion of the proposed studies will provide a new strategy to simultaneously inhibit three critical pathways via LIMK2, which lead to CRPC progression and enzalutamide-resistance. In vivo studies will reveal the potential of LIMK2 as a clinical target and LI-11 as a candidate drug. PDX mouse models with different LIMK2 levels will further uncover whether LIMK2 levels can serve as a novel predictive marker for determining the efficacy of CRPC therapy. The data further indicate that targeting LIMK2 could prevent the emergence of CRPC following androgen deprivation therapy. Finally, the validation of LIMK2 as a therapeutic target in CRPC may improve the odds of treating other aggressive cancers, where LIMK2 is upregulated.